Glycerol:NAD+2-OXIDOREDUCTASE (EC 1.1.1.6) was purified to homogeneity from a mutant of Escherichia coli K12 that uses this enzyme, instead of ATP:glycerol 3-phosphotransferase (EC 2.7.1.30), as the first enzyme for the dissimilation of glycerol. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit of 39,000 daltons. During electrophoresis under nondenaturing conditions, the protein migrates as two bands. These two forms, both of which are enzymatically active, appear to be dimers and octomers of the same subunit. The optimal pH for the oxidation of glycerol is about 10, and that for the reduction of dihydroxyacetone is about 6. Glycerol dehydrogenation is highly activated by NH4+, K+, or Rb+, but strongly inhibited by N-ethylmalemide, 8-hydroxyquinoline, 1,10-phenanthroline, Cu2+, and Ca2+. The enzyme exhibits a broad substrate specificity. In addition to glycerol, it act on 1,2-propanediol and several of its analogs.

At a concentration of 2.5 mM, DL-glyceraldehyde 3-phosphate has a bactericidal effect upon Escherichia coli. The glycerol 3-phosphate transport system is required for the entry of the biologically active L-enantiomer. L-Glyceraldehyde must be phosphorylated by the cell to exert itii, full effect upon growth. (diethyl acetal, monobarium salt); N,N-bis-(2-hydroxyethyl) glycine, Bicine; 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB; tris(hydroxymethyl)aminomethane; D-and L-glyceraldehyde; and octylphenoxypolyethoxyethanol, the nonionic detergent Triton X-100; and bovine serum albumin. D-Glyceraldehyde 3-phosphoric acid (diethyl acetal, dicyclohexyl ammonium salt) was a product of Boehringer Mannheim Corp., New York, N.Y. Glyceraldehyde 3-phosphoric acid was generated from the corresponding acetals by mixing with an aqueous suspension of Dowex 50, hydrogen ion form, using the procedure recommended by the Sigma Chemical Co.1-Palmitoyl glycerol 3-phosphate (lysophosphatidic acid) and cytidine diphosphate-dipalmitin (CDP-dipalmitin) were purchased from Serdary Re-

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Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation ofthe oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.

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